1. Field of the Invention
This invention relates to the synthesis and isolation of cDNA coding human lysozyme protein, the preparation of human lysozyme and human lysozyme protein itself. The invention also relates to the possibilities of therapeutic use thereof, as illustrated by way of example in the embodiments which follow. In particular, the invention relates to a bacterium plasmid which contains a cDNA sequence for part of the human lysozyme signal peptide, a cDNA sequence for the entire mature lysozyme protein and, at the 3' end, part of a non-coding sequence of the human lysozyme gene. The invention further relates to expression vectors such as plasmids, with the nucleotide sequences coding for human lysozyme as inserts and various host organisms or cultures which permit the preparation of human lysozyme.
2. Brief description of Background Information
Lysozymes are defined as 1,4-beta-N-acetylmuramidases which cleave the glycoside bond between the C-1 of N-acetyl-muramic acid (MurNAc) and the C-4 of N-acetylglucosamine (GlcNAc) in the peptidoglycan of bacteria (1). Alexander Fleming discovered in 1922 that various tissues and secretions as well as hen egg white were capable of lysing some gram positive bacteria. He called the lytic factor a lysozyme, i.e. an enzyme which is capable of lysing bacteria. Fleming showed that lysozyme occurs in homogenised tissue from cartilage and stomach, in tears, saliva, sputum, nasal secretions, pathological urine, serum and in leucocytes, but not in healthy urine, in the cerebrospinal fluid or in sweat (2). The activity of the lysozyme as an anti-bacterial agent appears to be based on both its direct bacteriolytic activity and also on stimulatory effects in connection with phagocytosis of macrophages (3,4).
The important role of lysozyme as a mediator in warding off microbes by means of alveolar macrophages has been demonstrated in the rat: in intact bacteria there is no phagocytosis, whereas lysozyme-damaged bacteria are rapidly phagocytosed (3). Similarly it has been shown that lysozyme can directly enhance the phagocytotic activity of polymorphonuclear leucocytes (5) and macrophages (4). Investigations have been carried out into the effect of lysozyme on microorganisms in the mouth, on seven serotypes of Streptococcus mutans, Veillonella alcalescens and virulent and non-virulent strains of Actinomyces viscosus T 14. The results showed that various mechanisms could be responsible for the bacteriostatic, lytic and bacteriocidal properties and that the enzyme is not only a selective factor but also an effective factor against microorganisms of the mouth (6). Other postulated functions of lysozyme include immune stimulation (7) and immunological and non-immunological monitoring of host membranes for any neoplastic transformation (8). Determination of the lysozymes from serum and/or urine is used to diagnose various diseases or as an indicator for their development. In acute lymphoblastic leukaemia the lysozyme serum level is significantly reduced, whereas in chronic myelotic leukaemia and in acute monoblastic and myelomonocytic leukaemia the lysozyme concentration in the serum is greatly increased (9,10).
The therapeutically effective use of lysozyme is possible in the treatment of various bacterial and virus infections (Zona, Herpes zoster), in colitis, various types of pain, in allergies, inflammation and in paediatrics (the conversion of cows milk into a form suitable for infants by the addition of lysozyme).
Lysozyme is able to interact with other biologically active proteins in such a way that they develop their full activity (Adinolfi, In: Lampert, Woods (eds), Academic Press, London, 1981, 19-47). Such components may be, for example, complement, lactotransferrin which inhibits the replication of certain microorganisms by forming iron-chelate complexes and antibodies such as sIgA in milk, which potentiates the antibacterial activity of lactotransferrin (Spik et al. Bull. Eur. Physiopath. Resp. 19, 123-130, 1983). Lysozyme, lactotransferrin and immunoglobulins also coexist in various natural secretions such as saliva, tears, various types of milk (Jorieux et al. Protides of the biological Fluids, Proc. 31st Coll. 1984), in the bronchial mucus membrane and in egg white. Lysozyme may additionally be used to advantage to alleviate rheumatic fever and rheumatic pain and has a therapeutic activity in diseases of the rheumatic or arthritic type (Third Int. Symp. on Flemings lysozyme, Milan 1964). Later, lysozyme was also credited with analgesic properties (Bianchi, Eur. J. Pharmacol. 71, 211-221, 1981). More recently, lysozyme was found to have an antinociceptive activity (Bianchi, Clin. Exp. Pharmacol. Physiol. 10, 45-52, 1983). This broad range of activities of lysozyme demonstrates a correspondingly large spectrum for therapeutic use, which means that it is of considerable economic importance.
In all the pharmaceutical applications of lysozyme enumerated here, human lysozyme (HLZ) is preferred to the lysozyme obtained from hen egg white since undesirable side effects of an anaphylactic and/or allergic nature are more likely when using lysozymes of a different species and could interfere with therapy.
Up till now, human milk and human placenta have been the only commercial sources for obtaining human lysozyme. However, the availability of these starting materials is very limited and it is obvious that different preparations will be obtained from one batch to the next. There ought to be advantages in using the widely developed industrial microbiology and the recently developed recombinant DNA technology to produce human lysozyme by means of microorganisms.
The gene for hen egg white lysozyme has been isolated (11,12) and the nucleotide sequence of hen egg white lysozyme mRNA and the exons located on the gene together with their flanking regions have been determined (13). Moreover, the nucleotide sequence of the lysozyme gene of bacteriophage T4 has been clarified (14). The amino acid sequence of HLZ (21,22,23) is known, but the nucleotide sequence coding for HLZ which is shown in this invention is not already known. EPA 181 634 describes the expression of human lysozyme in yeast and Bacillus subtilis. Apart from the fact that no special yeast terminator and no authentic HLZ gene is used in EPA 181 634, this HLZ DNA sequence has no preliminary DNA sequence coding for a leader peptide in the event of expression in yeast. The HLZ formed cannot therefore be transported from the host, with the result that it is difficult for an exact tertiary structure to be formed by corresponding disulphide bridge formation. There is therefore no description of how authentic HLZ is obtained according to EP-A181 634. Nor does it tell us how the start methionine is to be removed.